WO2007101925A1

WO2007101925A1 - Use of 3,5-seco-4-norcholestane derivatives for obtaining a cytoprotective medicament

Info

Publication number: WO2007101925A1
Application number: PCT/FR2007/000330
Authority: WO
Inventors: Rebecca Pruss; Bruno Buisson; Thierry Bordet
Original assignee: Trophos
Priority date: 2006-03-09
Filing date: 2007-02-23
Publication date: 2007-09-13
Also published as: KR20080102426A; CA2645086A1; FR2898272B1; BRPI0708657A2; US7985774B2; JP2009529517A; CN101437521B; HK1129583A1; CA2645086C; AU2007222282A1; EP1991235A1; US20090312434A1; KR101390018B1; AU2007222282B2; EP1991235B1; RU2008139988A; IL193774A0; JP5221392B2; RU2434635C2; MX2008011545A

Abstract

The present invention relates to the use of 3,5-seco-4-norcholestane derivatives for obtaining a cytoprotective medicament, with the exception of a neuroprotective medicament, in particular a cardioprotective or hepatoprotective medicament.

Description

Use of 3,5-seco-4-nor-cholestane derivatives for obtaining a cytoprotective medicament

The present invention relates to the use of 3,5-seco-4-norcholestane derivatives for obtaining a cytoprotective drug, with the exception of a neuroprotective drug.

Cellular degenerative processes are characterized by cell dysfunction often resulting in unwanted cell activities and cell death.

Cells have developed adaptation mechanisms, in response to stress, which lengthen their lifespan or delay or prevent cell death (cytoprotective mechanisms).

However, these cytoprotective mechanisms are sometimes insufficient, inadequate, or induced too late to be effective and cells die. It may therefore be interesting to have new drugs, cytoprotectors, which would promote cytoprotection. This is one of the aims of the present invention.

The term "cytoprotective" refers to the ability of agents, natural or not, to protect a cell against cell death, particularly pathological cell death, and / or against cellular dysfunctions leading to cell death. These cellular dysfunctions can for example be of mitochondrial origin, such as a reduction in the capacity to generate ATP, an inability to capture and / or retain calcium, or the generation of free radicals.

Among the main mechanisms of cell death, there are essentially necrosis, apoptosis and necroptosis.

Necrosis is a so-called "accidental" cell death that occurs during tissue damage. It is the plasma membrane of the cell that is most affected, causing a change in the homeostasis of the cell. The cells will soak up water to the point that it will cause the lysis of their plasma membrane. This cell lysis leads to the release into the surrounding medium of the cytoplasmic content. Necrosis is at the origin of the inflammatory process. The necrosis can affect a set of cells or a tissue while the other neighboring parts remain alive. The resulting transformation is a mortification of cells or tissues.

In other words, necrosis is defined by morphological changes occurring when a cell reaches the end of life following events such as a major trauma such as a stop or a decrease in blood circulation in an organ, hyperthermia (significant rise in temperature), poisoning by a chemical product, physical shock, etc. One of the most well-known necroses is that of the myocardium during infarction (arrest of circulatory supply to the muscle heart) due to obliteration (obstruction) of a coronary artery.

Apoptosis is an integral part of the normal physiology of an organism. It is a highly regulated physiological form of cell death and is necessary for the survival of multicellular organisms. Apoptosis is a process that plays a key role during embryogenesis.

The apoptotic or apoptotic cells will isolate themselves from the other cells. Apoptosis usually involves individual cells in a tissue and does not cause inflammation. One of the characteristic morphological points of apoptosis is the significant condensation of both the nucleus and the cytoplasm, which induces a significant decrease in cell volume. The nucleus then fragments, each fragment is surrounded by a double envelope. Apoptotic bodies (cytoplasmic and nuclear elements) are then released and will be absorbed by phagocytosis by neighboring cells.

Apoptosis can be induced in different ways. For example, radiation, the presence of a chemical compound or a hormone are stimuli capable of inducing a cascade of apoptotic events in the cell. Intracellular signals such as incomplete mitosis or damage to I ¹ DNA can also induce apoptosis.

Apoptosis also occurs after the action of a genotoxic agent or during an illness. Certain pathologies are characterized by an abnormal apoptosis, causing the loss of certain cell populations, such as for example hepatotoxicity, retinopathies, cardiotoxicity. A distinction is therefore made between physiological apoptosis and pathological apoptosis. The invention relates essentially to pathological apoptosis.

There are other mechanisms of cell death, such as necroptosis, which has characteristics of necrosis and apoptosis. A cell dying from necroptosis has characteristics similar to those of a cell dying from necrosis, but the biochemical stages of this mechanism are more similar to those of apoptosis. This cell death mechanism is involved, for example, in ischemia.

It is therefore also one of the aims of the present invention to have new drugs which could make it possible to prevent and / or treat necrosis and / or pathological apoptosis and / or necroptosis (antinecrotic and / or antiapoptotic drugs and / or antinecroptotics).

Cellular degenerative processes can result from, among other things, pathological situations grouped under the term of degenerative diseases or conditions, trauma, or exposure to various factors.

These traumas and factors can include, for example, exposure to radiation (UV, gamma), hypoxia or deprivation of oxygen, deprivation of nutrients, deprivation of growth factors, poisons, cellular toxins, waste, environmental toxins, free radicals, reactive oxygen or even certain medical events and / or procedures such as surgical trauma including transplants of cells, tissues and organs. Mention may also be made of chemical or biological agents used as therapeutic agents in the context of medical treatments such as, for example, cytostatic agents or anti-inflammatory agents.

The invention is not intended to treat the extracellular causes of pathologies or degenerative processes which can lead to cell death, but rather the consequences at the cellular level of said pathological processes or of said pathologies and in particular to protect the cell against said consequences. .

Among the most important pathological situations characterized by a degenerative process, other than neurological or neurodegenerative which the present invention does not address, there are: diseases of the bones, joints, connective tissue and cartilage, such as osteoporosis, osteomyelitis, arthritis including for example osteoarthritis, rheumatoid arthritis and psoriatic arthritis, avascular necrosis, progressive ossifying fibrodysplasia, rickets, Cushing's syndrome; muscle diseases such as muscular dystrophy, such as for example Duchenne muscular dystrophy, myotonic dystrophies, myopathies and myasthenia gravis; skin diseases, such as dermatitis, eczema, psoriasis, aging, or even scarring impairments; cardiovascular diseases such as cardiac and / or vascular ischemia, myocardial infarction, ischemic heart disease, chronic or acute heart failure, cardiac dysrhythmia, atrial fibrillation, ventricular fibrillation, paroxysmal tachycardia, heart failure, anoxia, hypoxia, side effects due to therapies with anticancer agents; circulatory diseases such as atherosclerosis, arterial sclerosis, and peripheral vascular diseases, strokes, aneurysms; hematological and vascular diseases such as: anemia, vascular amyloidosis, hemorrhages, sickle cell anemia, red cell fragmentation syndrome, neutropenia, leukopenia, aplastic anemia, pancytopenia, thrombocytopenia, l hemophilia; lung diseases including pneumonia, asthma; chronic obstructive lung diseases such as chronic bronchitis and emphysema; diseases of the gastrointestinal tract, such as ulcers; liver diseases such as hepatitis, particularly hepatitis of viral origin or having as causative agent other infectious agents, autoimmune hepatitis, fulminant hepatitis, certain hereditary metabolic disorders, Wilson's disease, cirrhosis, steatosis non-alcoholic liver, liver disease due to toxins or drugs; diseases of the pancreas such as, for example, acute or chronic pancreatitis; metabolic diseases such as diabetes mellitus and insipidus, thyroiditis; kidney diseases such as, for example, acute renal disorders or glomerulonephritis; viral and bacterial infections such as sepsis; severe poisoning from chemicals, toxins or drugs; degenerative conditions associated with Acquired Immune Deficiency Syndrome (AIDS); disorders associated with aging, such as accelerated aging syndrome; inflammatory diseases, such as Crohn's disease, rheumatoid arthritis; autoimmune diseases such as lupus erythematosus; dental disorders such as those leading to tissue degradation such as periodontitis; ophthalmic diseases or disorders including diabetic retinopathies, glaucoma, macular degeneration, retinal degeneration, retinitis pigmentosa, retinal holes or tears, retinal detachment, retinal ischemia, acute retinopathies associated with trauma, degenerations inflammatory, post surgical complications, drug retinopathies, cataracts; auditory pathway disorders, such as otosclerosis and deafness induced by antibiotics; diseases associated with mitochondria (mitochondrial pathologies), such as Friedrich's ataxia, congenital muscular dystrophy with structural mitochondrial anomaly, certain myopathies (MELAS syndrome, MERFF syndrome, Pearson syndrome), MIDD syndrome (mitochondrial diabetes) and deafness), Wolfram syndrome, dystonia. The invention also relates to the protection of cells, tissues and / or transplanted organs, whether before, during (removal, transport and / or reimplantation) or after transplantation.

We are always looking for pharmacologically active compounds to fight against the degenerative processes mentioned above.

The present invention responds to this demand for cytoprotective compounds. Indeed, the Applicant has discovered that the derivatives of 3,5-seco-4-norcholestane and in particular 3,5-seco-4-nor-cholestan-5-one oxime-3-ol and one of its esters are endowed with remarkable cytoprotective properties.

This is why the subject of the present invention is the use of at least one compound corresponding to formula I

in which

- X and Y taken together represent a keto function (= 0), an oxime group (= NOH) or a methyloxime group (≈NHOMe) where X represents a hydroxyl and Y a hydrogen atom,

- B represents a hydroxyl radical and C and D, identical or different, represent a hydrogen atom, or an alkyl radical, linear or branched, comprising from 1 to 4 carbon atoms, or B and C taken together represent a keto function and D a methyl, hydroxyl or methylamine radical, or B and C represent a hydrogen atom and D a methylamine radical, or B and C taken together represent an oxime group and D a methyl radical, and R represents an alkyl radical, linear or branched, comprising from 1 to 10 carbon atoms, or one of its addition salts with pharmaceutically acceptable acids, or one of its esters or one of the addition salts with pharmaceutically acceptable acids of said esters, for the preparation of a cytoprotective medicament, with except for a neuroprotective drug.

According to the invention, the addition salts with pharmaceutically acceptable acids can be, for example, salts formed with hydrochloric, hydrobromic, nitric, sulfuric, phosphoric, acetic, formic, propionic, benzoic, maleic, fumaric, succinic, tartaric acids. , citric, oxalic, glyoxylic, aspartic, alkane sulfonic such as methane or ethane sulfonic, arylsulfonic, such as benzene or paratoluene sulfonic, or carboxylic acids.

According to the invention, the oxime group represents the pure and mixed syn and anti isomers associated with the orientation of the N-O bond with respect to the double bond C = N.

According to a particular form of the invention, the radical R of the compounds of formula I which can be used is preferably the cholestane radical of formula II

According to other particular forms of the invention, the compounds of formula I for which X and Y taken together represent a keto function or represent an oxime group, are preferably used.

According to yet other particular forms of the invention, the compounds of formula I for which B represents a hydroxyl radical and C and D, identical or different, represent a hydrogen atom or an alkyl radical, linear or branched, comprising 1 to 4 carbon atoms, or for which B and C taken together represent a keto function and D represents a methyl radical, are preferably used.

Very preferably, at least one compound of formula I chosen from 3,5-seco-4-nor-cholestan-5-one oxime-3-ol is used according to the invention, 3,5-seco-4-nor-cholestan-5-one oxime-3-methyl alcohol, or 3,5-seco-4-nor-cholestan-5-one oxime-3-dimethyl alcohol, or one of its addition salts with pharmaceutically acceptable acids, or one of its esters or one of the addition salts with pharmaceutically acceptable acids of said esters.

The interesting cytoprotective properties of the compounds of formula I justify their use for the preparation of a cytoprotective drug, particularly intended for the treatment or prevention of necrosis and / or apoptosis and / or necroptosis (antinecrotic drugs and / or antiapoptotics and / or antinecroptotics) or diseases such as bone, joint, connective tissue and cartilage diseases, muscle diseases, skin diseases, cardiovascular diseases, circulatory diseases, hematological and vascular diseases , lung disease, gastrointestinal tract disease, liver disease, pancreatic disease, metabolic disease, kidney disease, viral and bacterial infections, severe poisoning, degenerative conditions associated with Immuno Syndrome Acquired deficit (AIDS), disorders associated with aging, inflammatory diseases, autoimmune diseases, dental disorders, ophthalmic diseases or disorders, diseases of the auditory tract, diseases associated with mitochondria (mitochondrial pathologies). The invention also relates to the protection of cells, tissues or transplanted organs, whether before, during (removal, transport and / or reimplantation) or after transplantation.

Advantageously, the compounds of formula I can be used in the preparation of a medicament intended for the protection of cardiac cells (cardioprotective medicament), in the protection of hepatic cells (hepatoprotective medicament) or of a medicament intended for the treatment or prevention of diseases associated with mitochondria.

According to the invention, the compound of formula I is advantageously present in the cytoprotective medicament at physiologically effective doses; said drugs contain in particular an effective cytoprotective dose of at least one of the compounds of formula I.

As medicaments, the compounds corresponding to formula I, their esters, their addition salts with pharmaceutically acceptable acids as well as the addition salts with pharmaceutically acceptable acids of said esters can be formulated for the digestive or parenteral route.

The medicaments according to the invention may also comprise at least one other therapeutically active ingredient, whether it is active on the same pathology or on a different pathology, for simultaneous, separate or spread over time use, in particular during a treatment in a subject suffering from one of the pathologies previously mentioned.

According to the invention, the compound of formula I can be used in the medicament, in mixture with one or more excipients or inert vehicles, ie pharmaceutically inactive and non-toxic. Mention may be made, for example, of saline, physiological, isotonic, buffered solutions, etc., compatible with pharmaceutical use and known to those skilled in the art. The compositions may contain one or more agents or vehicles chosen from dispersants, solubilizers, stabilizers, preservatives, etc. Agents or vehicles usable in formulations (liquids and / or injectables and / or solids) are in particular methylcellulose, hydroxymethylcellulose, carboxymethylcellulose, cyclodextrins, polysorbate 80, mannitol, gelatin, lactose, vegetable oils or animal, acacia, etc. The compositions can be formulated in the form of an injectable suspension, gels, oils, tablets, suppositories, powders, capsules, capsules, etc., optionally by means of dosage forms or devices ensuring prolonged and / or delayed release. For this type of formulation, an agent such as cellulose, carbonates or starches is advantageously used.

The administration can be carried out by any method known to a person skilled in the art, preferably by oral route or by injection, typically by intraperitoneal, intra-cerebral, intra-thecal, intravenous, intra-arterial or intra route. -muscular. Oral administration is preferred. For long-term treatment, the preferred route of administration will be sublingual, oral or transcutaneous.

For injections, the compounds are generally packaged in the form of liquid suspensions, which can be injected using syringes or infusions, for example. It is understood that the flow rate and / or the dose injected, or in general the dose to be administered, can be adapted by a person skilled in the art according to the patient, the pathology, the mode of administration, etc. it is understood that repeated administrations can be carried out, optionally in combination with other active ingredients and / or any pharmaceutically acceptable vehicle (buffers, saline, isotonic solutions, in the presence of stabilizing agents, etc.).

The invention can be used in mammals, in particular in humans.

In general, the daily dose of the compound will be the minimum dose to obtain the desired therapeutic effect. The doses of the compounds described above and for example 3,5-seco-4-nor-cholestan-5-one oxime-3-ol will generally be between 0.001 to 100 mg per kilo per day for humans.

If necessary, the daily dose can be administered in two, three, four, five, six or more, taken per day or in multiple sub-doses administered at appropriate intervals during the day.

The amount chosen will depend on multiple factors, in particular the route of administration, the duration of administration, the time of administration, the rate of elimination of the compound, or the different product (s) used in combination with the compound, age, weight and physical condition of the patient, as well as his medical history, and any other information known in medicine.

The doctor's prescription may start at doses lower than those generally used, then these doses will be gradually increased in order to better control the appearance of possible side effects.

The compositions according to the invention, in particular the pharmaceutical or medicinal compositions, can comprise at least one compound of formula I as described above, or one of its addition salts with pharmaceutically acceptable acids, or one of its esters or one of the addition salts with pharmaceutically acceptable acids of said esters,

The pharmaceutical compositions according to the invention may also comprise at least one other therapeutically active ingredient, for simultaneous, separate or spread over time use, in particular during treatment in a subject suffering from one of the pathologies mentioned above.

The pharmaceutical compositions according to the invention can advantageously comprise one or more inert excipients or vehicles, that is to say pharmaceutically inactive and non-toxic.

The compounds of formula I used according to the invention can be synthesized by reacting a compound of formula III

in which R has the meanings indicated above which are subjected to either the action of methylamine and then hydroxylamine to obtain a compound of formula I in which R has the meanings previously indicated, X and Y together represent a group oxime, B represents together with C a keto function and D a methyl amino group - either to methylation to obtain a compound of formula IV

in which R has the meanings previously indicated, which are subjected to the action of an agent for protecting the ketone function in position 5 in order to obtain a compound of formula V

in which R has the meanings previously indicated, that

- Or else it is reacted with methyl lithium and then subjected to the action of an agent for deprotecting the ketone function in position 5, then it is reacted with hydroxylamine to obtain a compound of formula I in which R has the meanings previously indicated, X and Y together represent an oxime group, B represents a hydroxyl group and C and D represent linear or branched alkyl radicals of 1 to 4 carbon atoms,

- Or saponify, then react with a compound of formula H ₃ C-NH-OCH _3, then react with methyl lithium, to obtain a compound of formula Vl

which is subjected to a reduction in the ketone function then it is subjected to the action of an agent for deprotecting the ketone function in position 5 then it is reacted with the hydroxylamine to obtain a compound of formula I in which R has the meanings previously indicated, X and Y together represent an oxime group, B represents a hydroxyl group, and C represents a linear or branched alkyl radical of 1 to 4 carbon atoms optionally substituted and D represents an atom of hydrogen,

- or else it is reduced to obtain a compound of formula VII

in which R has the meanings previously indicated, B represents a hydroxyl group, and C and D represent a hydrogen atom, that

- either subject to the action of an oxidizing agent to obtain a compound of formula VIII

(VIII) in which R has the meanings previously indicated, the Schiff base of which is prepared and then reduced, then subjected to the action of an agent for deprotecting the ketone function in position 5, then reacting with the hydroxylamine to obtain a compound of formula I in which R has the meanings previously indicated, X and Y together represent an oxime group, B represents a methylamine group, and C and D represent a hydrogen atom,

- either subject to the action of an agent for deprotecting the ketone function in position 5, then reacting with an amine chosen from hydroxylamine, methylhydroxylamine and carboxymethylhydroxylamine to obtain a compound of formula I in which R has the meanings indicated above, X and Y together respectively represent an oxime, methyloxime, and carboxymethyloxime group, B represents a hydroxyl group, and C and D represent a hydrogen atom, and it is isolated and if desired salifies the compounds of formula I or they are esterified, isolated and then if desired salifies them.

Under preferential conditions for implementing the process described above,

- the reaction of the compound of formula III with methylamine is carried out in the presence of a coupling agent activating the acid function such as BOP (Benzotriazole-i-yl-oxy-tris- (dimethylamino) -phosphonium hexafluorophosphate) or TBTU ( 2- (1H-benzotriazole-1-yl) -1, 1, 3,3-tetramethyluronium tetrafluoroborate) advantageously in the presence of a base such as N-methylmorpholine, in particular in a suitable solvent such as dichloromethane or dimethylformamide. Preferably, it is carried out in the presence of EDCI (1-Ethyl-3- (3'-dimethylaminopropyl) carbodiimide) combined with 4-dimethylaminopyridine in dichloromethane, the mixture being stirred at room temperature for 24 h. The product is then preferably dissolved in pyridine, then 5 to 7 and in particular 6 equivalents of hydroxylamine hydrochloride are added.

the methylation of the compound of formula III is carried out by reaction with methanol in the presence of thionyl chloride, preferably by solubilizing the acid of formula II in a suitable volume of a mixture of 70% methanol and 30% dichloromethane. The mixture is cooled to 0 ^° C. and 3 equivalents of thionyl chloride are added dropwise. Then stirred for 2 hours at room temperature. On this compound, the protection of the ketone function is preferably carried out by dissolving the product in an excess, for example 10 equivalents of trimethylorthoformate and a sufficient volume of ethylene glycol, then addition of anhydrous p-toluenesulfonic acid.

- The reaction of the compound of formula V with methyl lithium is preferably carried out in anhydrous THF then after cooling to about - 45 ⁰ C, dropwise addition of an excess of methyl lithium. Deprotection of the dioxolane which blocks the ketone function in position 5, is carried out in acetone in the presence of sulfuric acid. Preferably it is carried out in dioxane, in the presence of a water / acetic acid 1/1 mixture. The ketone oxime is advantageously carried out as above.

- The saponification of the compound of formula V is carried out with sodium hydroxide, preferably in dioxane. About 2 equivalents of an aqueous sodium hydroxide solution are added in particular. This product is reacted with a compound of formula H ₃ C-NH-OCH ₃ for example in the presence of a coupling agent activating the acid function such as BOP or TBTU in the presence of a base such as N-methylmorpholine in a suitable solvent such as dichloromethane or dimethylformamide. Preferably, it is carried out in the presence of EDCI associated with hydroxybenzotriazole with triethylamine added dropwise in the solvent. This product is reacted with methyl lithium under an argon atmosphere according to the protocol described above, then the ketone function in 3 is reduced by sodium borohydride. The product obtained is then subjected to the deprotection of the ketone function in position 5 and reacted with the hydroxylamine according to the same protocol described above.

- The reduction of the compound of formula V to obtain the compound of formula VII is preferably carried out with lithium aluminum hydride in particular by placing it in suspension in tetrahydrofuran. It is carefully hydrolyzed by the addition of a sodium sulphate solution.

- The oxidation of the compound of formula VII is carried out using pyridimium chlorochromate. On this product we obtain the basis of Schiff which is instantly reduced especially by dissolving under argon preferably in ethanol in the presence of triethylamine, methylamine hydrochloride and titanium tetraisopropoxide, then addition of sodium borohydride. The deprotection of the ketone function in position 5 as well as the reaction with the hydroxylamine are carried out under the conditions previously described.

The examples which follow illustrate the present application without however limiting it.

Examples

The retention times below are expressed in minutes and hundredths of a minute.

The liquid chromatography method used for all of the products is as follows:

Column: Macherey-Nagel - Nucleosil® 300-6 C4 - 150x4.6 mm Gradient: water (+ 0.05% trifluoroacetic acid) / acetonitrile (+ 0.05% trifluoroacetic acid) t = 0 min: 60% acetonitrile, 40% H ₂ O t = 6 min: 100% acetonitrile, 0% H ₂ O t = 11 min: 100% acetonitrile, 0% H ₂ O t = 13 min: 60% acetonitrile, 40% H ₂ O t = 15 min: 60 % acetonitrile, 40% H ₂ O.

The ionization conditions for the mass spectrometer are:

Source temperature: 25O ⁰ C

Cone voltage: 50 V

Capillary voltage: 3 kV

Rf lens: 0.3 V

Example 1: Synthesis of 3,5-seco-4-nor-cholestane-5-one, oxime-3-methylamide

Step A: Firstly, 250 mg of 3,5-seco-4-nor-cholestane-5-one, oxime-3-oic acid, 38 mg of methylamine hydrochloride, 250 mg of EDCI, 100 mg of DMAP and 2.5 mL of dichloromethane. The solution is stirred for 24 hours at room temperature then the reaction medium is diluted by adding dichloromethane and washed with a 10% sodium bicarbonate solution. The organic phase is dried over sulphate magnesium then concentrated under reduced pressure. The residue obtained is purified by flash chromatography (CH ₂ CI ₂ / MeOH 95/5). 176 mg of 3,5-seco-4-nor-cholestane-5-one-3-methylamide are recovered with a yield of 68%.

Analysis

1 H NMR (CDCI3): compliant

Retention time: 4 min 42 hundredths

Peaks detected in mass spectrometry: [M + H] + = 418; [2M + H] + = 835

Step B: Next, 50 mg of 3,5-seco-4-norcholestane-5-one-3-methylamide, 50 mg of hydroxylamine hydrochloride in 1 ml of pyridine are introduced into a flask. The mixture is stirred for 16 hours at ambient temperature then the reaction medium is concentrated under reduced pressure. The residue obtained is taken up in a CH2Cl2 / H ₂ O mixture; the organic phase is separated, washed with water, dried over anhydrous sodium sulfate and concentrated under reduced pressure. 40.6 mg of 3,5-seco-4-nor-cholestane-5-one, oxime-3-methylamide are recovered with a yield of 78%.

Analysis

1 H NMR (CDCI ₃ ): compliant

Retention time: 3 min 70 hundredths

Peaks detected in mass spectrometry: [M + H] + = 433; [2M + H] + = 865

Example 2 Synthesis of 3,5-seco-4-nor-cholestane-5-one-3-dimethyl alcohol

Step A: In a flask, 10.5 g of 3,5-seco-4-nor-cholestane-5-one-3-oic acid are dissolved in 378 ml of methanol and 146 ml of dichloromethane. It is cooled to 0 ^° C. and 5.7 ml of thionyl chloride are added dropwise. Then stirred for 2 hours at room temperature. The reaction medium is concentrated under reduced pressure, co-evaporated with toluene and then with dichloromethane. 10.3 g of 3,5-seco-4-nor-cholestane-5-one-3-methyl ester are obtained with a yield of 94%. The product is used as is without purification.

1 H NMR (CDCI ₃ ): compliant

Retention time: 4 min 69 hundredths

Peak detected in mass spectrometry: {M + H} + = 419; [2M + H] + = 785

Step B: In a flask, 9.62 g of 3,5-seco-4-nor-cholestane-5-one-3-methyl ester are dissolved in 25 ml of trimethylorthoformate and 53 ml ethylene glycol. 400 mg (2.3 mmol) of anhydrous p-toluenesulfonic acid are then added and the mixture is then stirred overnight at room temperature. Ethyl acetate is added to the reaction medium; washing is carried out with a 10% solution of sodium hydrogencarbonate. The organic phase is separated, dried over anhydrous magnesium sulfate and concentrated under reduced pressure. 9.95 g of 3,5-seco-4-nor-cholestane-5,5 (ethylene dioxy) -3-methyl ester are obtained with a yield of 93%. The product is used as is without purification.

1 H NMR (CDCI ₃ ): compliant

Retention time: 5 min 76 hundredths

Peak detected in mass spectrometry: {M + H} + = 463

Step C: In a flask, 300 mg of 3,5-seco-4-norcholestane-5,5 (ethylene dioxy) -3-methyl ester are dissolved in 5 ml of anhydrous THF. The medium is cooled to -45 ^° C. then 1.36 ml of a 1.6M solution of methyllithium in ether is added dropwise. After 30 minutes of stirring at -45 ° C. A few drops of methanol are added to the reaction medium and brought to room temperature. It is taken up in 20 ml of diethyl ether and washed with a saturated solution of sodium bicarbonate, then with a saturated solution of sodium chloride. The organic phase is dried over magnesium sulfate, then concentrated under reduced pressure. 295 mg is obtained 3,5-seco-4-nor-cholestane-5,5 (ethylene dioxy) -3-dimethyl alcohol (MW = 462) with a yield of 98%.

Retention time: 5 min 56 hundredths

Peaks detected in mass spectrometry: [M- (CH ₂ OH-CH ₂ OH + H ₂ O) + H] +] = 401

Step D: 6 ml of a 1/1 water / acetic acid mixture and 295 mg of 3,5-seco-4-nor-cholestane-5,5 (ethylene dioxy) -3-dimethyl alcohol are added to a flask ; heated at reflux for 1 hour 30 minutes. After cooling, the reaction medium is diluted with ethyl acetate, washed with a saturated solution of sodium chloride, then with a saturated solution of sodium bicarbonate. Finally, the organic phase is dried over magnesium sulfate and concentrated under reduced pressure. The crude product obtained is purified by flash chromatography (petroleum ether / ethyl acetate 8/2). 180 mg of 3,5-seco-4-nor-cholestane-5-one-3-dimethyl alcohol are obtained with a yield of 68%. 1 H NMR (CDCI ₃ ): compliant Retention time: 5 min 08 hundredths

Peaks detected in mass spectrometry: [M + H] + = 419; [M- H2O + H] + = 401; [2M + H] + = 837

Example 3 Synthesis of 3,5-seco-4-nor-cholestane-5-one, oxime-3-dimethyl alcohol

1 g of the compound of Example 2, 1 g of hydroxylamine hydrochloride in 53 ml of pyridine and a few ml of dichloromethane are introduced into a flask to dissolve the ketone. The mixture is stirred for 16 hours at ambient temperature then the reaction medium is concentrated under reduced pressure. The residue obtained is taken up in a CH ₂ CI ₂ / H ₂ O mixture; the organic phase is separated, washed with water, dried over anhydrous sodium sulfate and concentrated under reduced pressure. 814 mg of 3,5-seco-4-nor-cholestane-5-one, oxime-3-dimethyl alcohol are recovered with a yield of 78%. 1 H NMR (CDCI ₃ ): compliant Retention time: 5 min 09 hundredths

Peaks detected in mass spectrometry: [M + H] + = 434; [2M + H] + = 867 Example 4: synthesis of 3,5-seco-4-nor-cholestane-5-one, oxime-3-methyl alcohol

Step A: In a flask, 2 g of 3,5-seco-4-nor-cholestane-5,5 (ethylene dioxy) -3-methyl ester are placed in 26 ml of dioxane. 8.6 ml of a 1N sodium hydroxide solution are added. The reaction medium is heated at reflux for 1 hour 30 minutes and the dioxane is evaporated under reduced pressure. The solution obtained is acidified by adding a 1N hydrochloric acid solution to pH = 1 and extracted twice with toluene. The organic phases are combined, dried over anhydrous magnesium sulfate and concentrated under reduced pressure. 1.92 g of 3,5-seco-4-nor-cholestane-5,5 (ethylene dioxy) -3-oic acid are recovered with a yield of 99% which is used without additional treatment in the following step.

Step B: In a flask, 1.9 g of 3,5-seco-4-nor-cholestane-5,5 (ethylene dioxy) -3-oic acid are placed in 30 ml of dichloromethane. 1.06 g of EDCI, 743 mg of HOBT, 537 mg of N ₁ O-dimethylhydroxylamine hydrochloride and then 1.37 ml of triethylamine are added dropwise to this solution. We shake at room temperature for 16 hours. A CH ₂ CI ₂ ZH ₂ O mixture is added to the reaction medium and it is extracted 3 times with dichloromethane. The organic phases are combined, dried over anhydrous magnesium sulfate and concentrated under reduced pressure. The residue obtained is purified by flash chromatography (CHaCb / ethyl acetate 8/2). Recovering 1.46 g 3,5-seco-4-nor-cholestane-5,5 (ethylene dioxy) -3- (N, N-methoxy-methyl) amide with a yield of 70%.

1 H NMR (CDCI ₃ ): compliant

Retention time: 5 min 31 hundredths

Peak detected in mass spectrometry: [M + H] + = 492

Step C: 1.4 g of 3,5-seco-4-nor-cholestane-5,5 (ethylene dioxy) -3- (N, N-methoxy-methyl) amide are introduced into a balloon under argon. mL of anhydrous tetrahydrofuran and the mixture is cooled to 0 ^° C. 3.38 mL of a 1.6M solution of methyllithium in ether is then added dropwise. The reaction medium is stirred for 3 hours 40 minutes at 0 ^° C. and then a solution of 0.72 ml of concentrated hydrochloric acid in 7.28 ml of water is added dropwise. The tetrahydrofuran is evaporated under reduced pressure; the aqueous solution obtained is basified by adding 1 N sodium hydroxide solution to pH = 10. Extraction is carried out with diethyl ether; the organic phases are combined, dried over anhydrous magnesium sulfate, and concentrated under reduced pressure. The residue obtained is purified by flash chromatography (petroleum ether / ethyl acetate 9/1). 930 mg of 3,5-seco-4-nor-cholestane-5,5 (ethylene dioxy) -3-methyl ketone are recovered with a yield of 73%.

1 H NMR (CDCI ₃ ): compliant

Retention time: 5 min 65 hundredths

Peak detected in mass spectrometry: [M + H] + = 403

Step D: In a flask, 119 mg of 3,5-seco-4-nor-cholestane-5,5 (ethylene dioxy) -3-methyl ketone from step C are placed in 1.5 ml of methanol. Cool to 0 ° C and add 10 mg of sodium borohydride. The reaction medium is stirred at 0 ^° C. for 1 h and then concentrated under reduced pressure. The residue is taken up in water and extracted with dichloromethane. The organic phase is dried over magnesium sulfate and concentrated under reduced pressure. We recovers 94 mg 3,5-seco-4-nor-cholestane-5,5 (ethylene dioxy) -3-methylalcohol with a yield of 78%, this product is used as it is. 1 H NMR (CDCI ₃ ): compliant Retention time: 5 min 22 hundredths Peaks detected in mass spectrometry: [M + H] + = 387 Step E: The operation is carried out as in step D of Example 2 to deprotect the ketone in 5.

Step F: 121 mg of 3,5-seco-4-nor-cholestane-5-one-3-methyl alcohol, 1.5 ml of pyridine and 121 mg of hydroxylamine hydrochloride are introduced into a flask. The solution is stirred for two days at room temperature. The reaction medium is concentrated under reduced pressure, taken up in water and extracted with dichloromethane. The organic phase is then washed with water, then dried over magnesium sulfate and concentrated under reduced pressure. The product thus obtained is purified by flash chromatography (petroleum ether / ethyl acetate 9/1). 66 mg of 3,5-seco-4-nor-cholestane-5-one, oxime-3-methyl alcohol are obtained with a yield of 53%. 1 H NMR (CDCI ₃ ): compliant Retention time: 4 min 91 hundredths Peaks detected in mass spectrometry: [M + H] + = 420 Example 5: synthesis of 3,5-seco-4-nor-cholestane- 5-one, oxime-3-methylamine

Step A: In a balloon, 615 mg of LiAIH4 is placed in suspension in 57 ml of THF. It is cooled to 0 ^° C. and a solution of 3.0 g of 3,5-seco-4-nor-cholestane-5,5 (ethylene dioxy) -3-methyl ester is added dropwise. 57 mL of tetrahydrofuran. Then stirred at 0 ^° C. for 5 h. It is carefully hydrolyzed by adding a solution of sodium sulfate; the white solution obtained is stirred for 30 minutes, then filtered. The filtrate is concentrated under reduced pressure and taken up in water, extracted with ethyl acetate. The organic phase is dried over magnesium sulfate and then concentrated under reduced pressure. 2.55 g is obtained 3.5-seco-4-nor-cholestane-5.5 (ethylene dioxy) -3-ol with a yield of 85%, this product is used as it is. 1 H NMR (CDCI ₃ ): compliant Retention time: 4 min 82 hundredths Peaks detected in mass spectrometry: [M- (CH2OH-CH2OH + H2O) + H] + = 373

Step B: In an argon flask, 476 mg of 3,5-seco-4-nor-cholestane-5.5 (ethylene dioxy) -3-ol are dissolved in 7 ml of dichloromethane, then 189 mg of d neutral alumina and 399 mg of pyridinium chlorochromate; stirred at room temperature for 3:30. The reaction medium is filtered through Celite®; the filtrate is concentrated under reduced pressure. The residue obtained is purified by flash chromatography (toluene / ethyl acetate 9/1 then 8/2). 328 mg of 3,5-seco-4-nor-cholestane-5,5 (ethylene dioxy) -3-al are obtained with a yield of 69%.

Retention time: 5 min 57 hundredths Peaks detected in mass spectrometry: [M + H] + = 433 Step C: In a flask under argon, 323 mg of 3,5-seco-4-nor-cholestane-5 is solubilized , 5 (ethylene dioxy) -3-al in 3 mL of ethanol, then 209 μL of triethylamine, 100 mg of methylamine hydrochloride and 444 μL of titanium tetraisopropoxide are added. The reaction medium is stirred for 6 h at room temperature, then 42.5 mg of sodium borohydride are added. The mixture is stirred for 16 hours at room temperature. The reaction medium is filtered and washed with dichloromethane. The filtrate is dried over magnesium sulfate and concentrated under reduced pressure. The residue obtained is purified by flash chromatography (dichloromethane / methanol 9/1 to 5/5). 84 mg of 3,5-seco-4-nor-cholestane-5,5 (ethylene dioxy) -oxime-3-methylamine are obtained with a yield of 25%.

1 H NMR (CDCI ₃ ): compliant Retention time: 3 min 93 hundredths Peaks detected in mass spectrometry: [M + H] + = 448 Step D: 50 mg of 3,5-seco- are introduced into a flask 4-nor-cholestane- 5,5 (ethylene dioxy) -3-methylamine and 976 μL of a water / acetic acid 1/1 mixture. The mixture is brought to reflux for 6 hours. After cooling, the reaction medium is diluted with ethyl acetate and washed with a saturated sodium chloride solution and then with a 5% sodium bicarbonate solution. The organic phase is dried over magnesium sulfate and concentrated under reduced pressure. The product obtained is purified by flash chromatography (dichloromethane / methanol 95/5); 5 mg 3,5-seco-4-nor-cholestane-5-one-3-methylamine is obtained with a yield of 11%. 1 H NMR (CDCI ₃ ): compliant Retention time: 3 min 68 hundredths Peaks detected in mass spectrometry: [M + H] + = 404 Step E: 5 mg of 3,5-seco- are introduced into a flask 4-nor-cholestane-5-one-3-methylamine, 5 mg of hydroxylamine hydrochloride and 287 μl of pyridine. The mixture is stirred for 16 hours at room temperature. It is then taken up in dichloromethane and washed with water. The organic phase is dried over magnesium sulfate and concentrated under reduced pressure. 5 mg of 3,5-seco-4-nor-cholestane-5-one, oxime-3-methylamine are obtained with a yield of 91%.

Retention time: 3 min 66 hundredths Peaks detected in mass spectrometry: [M + H] + = 419 Example 6: synthesis of 3,5-seco-4-nor-cholestane-5-one, methyloxime-3-ol

20 mg of 3,5-seco-4-nor-cholestane-5-one-3-ol, 20 mg of O-methylhydroxylamine hydrochloride in 1 ml of pyridine are introduced into a flask. Agitation is carried out for 36 h at room temperature and 10 mg of O-methylhydroxylamine hydrochloride are added. The mixture is again stirred for 16 hours at ambient temperature then the reaction medium is concentrated under reduced pressure. The residue obtained is taken up in a CH ₂ CI ₂ / H ₂ O mixture; the organic phase is separated, washed with water, dried over anhydrous magnesium sulfate and concentrated under reduced pressure. 18 mg of a yellow oil are obtained which is purified by flash chromatography (petroleum ether / ethyl acetate 9/1). 5.8 mg of 3,5-seco-4-nor-cholestane-5-one, methyloxime-3-ol are recovered with a yield of 27%. Analysis

1 H NMR (CDCI ₃ ): compliant Retention time: 5 min 50 hundredths Peak detected in mass spectrometry: [M + H] + = 420 EXAMPLE 7 Synthesis of 3,5-seco-4-nor-cholestane-5-one carboxymethyIoxime-3-ol

52 mg of ketone, 25 mg of carboxymethoxylamine hemi-hydrochloride in 0.5 ml of pyridine are introduced into a flask. The mixture is stirred for 2 days at ambient temperature then the reaction medium is concentrated under reduced pressure. The residue obtained is taken up in a CH ₂ CI ₂ / H ₂ O mixture; the organic phase is separated, washed with water and then with a 2% hydrochloric acid solution, dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue obtained is purified by flash chromatography (petroleum ether / ethyl acetate 8/2). 24 mg are obtained with a yield of 39% of the carboxymethyloxime.

Analysis

1 H NMR (CDCI ₃ ): compliant

Retention time: 4 min 40 hundredths

Peak detected in mass spectrometry: [M + H] + = 464; [2M + H] ⁺ = 927

Example 8

A suspension corresponding to the formula has been prepared

3,5-seco-4-nor-cholestan-5-one oxime- 3-ol 20 mg per ml

Excipient: Oily emulsion

Example 9

We prepared a dry form corresponding to the formula

3,5-seco-4-nor-cholestan-5-one oxime- 3- N.N-dimethylglycine ester 250 mg hydrochloride

Excipient: qsp one 750 mg capsule finished

Example 10 Synthesis of 3,5-seco-4-nor-cholestan-5-one oxime-3- N, N-dimethylglycine ester: prodrug of 3,5-seco-4-nor-choIestan-5-one oxime- 3-ol

In a flask, 509 mg of 3,5-seco-4-nor-cholestan-5-one-3-ol, 182 mg of N hydrochloride, N-dimethylglycine, 275 mg of EDCI and 207 mg of DMAP are placed in 10 to 15 mL of dichloromethane. The mixture is stirred at room temperature for 16 hours. A 5% sodium bicarbonate solution is added to the reaction medium and the mixture is extracted with dichloromethane. The organic phases are combined, dried over anhydrous sodium sulfate and concentrated under pressure scaled down. The residue obtained is purified by flash chromatography (toluene / ethyl acetate 8/2). 488 mg are recovered with a yield of 78%.

Analysis

1 H NMR (CDCI ₃ ): compliant

Retention time: 3 min 77 hundredths

Peak detected in mass spectrometry: [M + H] + = 476

The product is then used in the following reaction:

488 mg of the product obtained and 488 mg of hydroxylamine hydrochloride in 23 ml of pyridine are introduced into a flask. The mixture is stirred for 16 hours at ambient temperature and then the reaction medium is taken up in a CH2Cl ₂ / H ₂ O mixture; Ia organic phase is separated, washed with water, dried over anhydrous sodium sulfate and concentrated under reduced pressure. 378 mg of the oxime are recovered with a yield of 75%. The product is then salified in the presence of an ether solution acidified with an HCl solution, to obtain the product in the form of the hydrochloride.

Analysis

1 H NMR (CDCI ₃ ): compliant

Retention time: 3 min 43 hundredths

Peak detected in mass spectrometry: [M + H] + = 491

Example 11 Synthesis of 3,5-seco-4-nor-cholestan-5-one, oxime-3- (4-methyl-1-piperazine) propanoate ester: Prodrug of 3,5-seco-4-nor-cholestan -5-one, oxime-3-ol

264 mg of 3,5-seco-4-nor-cholestan-5-one-3-ol, 121 mg of 4-methyl-1-piperazine propanoic acid in the form of a lithium salt are placed in a flask, 1425 mg EDCI and 106 mg DMAP in 2 to 3 mL of dichloromethane. The mixture is stirred at room temperature for 1 night. Water is added to the reaction medium and the mixture is extracted with dichloromethane. The organic phases are combined, dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue obtained is purified by flash chromatography (toluene / ethyl acetate 98/2). 54 mg of the desired product are recovered with a yield of 15%.

Analysis

1 H NMR (CDCI ₃ ): compliant

Retention time: 3 min 66 hundredths Peak detected in mass spectrometry: [M + H] + = 545

The product is then used in the following reaction:

30 mg of the product obtained and 30 mg of hydroxylamine hydrochloride in 1.2 ml of pyridine are introduced into a flask. Stirred 5:30 at room temperature then the reaction medium is taken up in a CH ₂ CI ₂ / H ₂ O mixture; the organic phase is separated, washed with water, dried over anhydrous sodium sulfate and concentrated under reduced pressure. 19 mg of the oxime are recovered with a yield of 13%.

Analysis

1 H NMR (CDCI ₃ ): compliant

Retention time: 3 min 62 hundredths

Peak detected in mass spectrometry: [M + H] + = 560

The product is then salified in the presence of an ether solution acidified with an aqueous hydrochloric acid solution, to obtain the product in the form of di-hydrochloride.

Example 12 Anti-apoptotic effect of 3,5-seco-4-nor-cholestan-5-one oxime-3-ol: Contractility and apoptosis of rabbit ventricular cardiomyocytes

The antiapoptotic properties of 3,5-seco-4-nor-cholestan-5-one oxime-3-ol (Azasteroidal alkaloids. Synthesis of A-nor-B-homo-5-azacholestane. Rodewald, WJ; Wicha, J. Univ. Warsaw, Bulletin of the Polish Academy of Sciences, Series of Chemical Sciences (1963), 11 (8), 437-441) were analyzed on cardiomyocytes, by a contractile dysfunction test induced by doxorubicin. > Materials and Methods

Compound to be tested

A stock solution of 3,5-seco-4-nor-cholestan-5-one oxime-3-ol at a concentration of 10 mM in 100% DMSO was used.

The final concentration of DMSO was the same for all the experimental points, independently of the concentrations of molecules used.

3,5-seco-4-nor-cholestan-5-one oxime-3-ol was tested at concentrations of 0.1 and 0.3 μM, diluted in a solution of Tyrode (composition in mmol / L: NaC1 135, KCL 5.4, NaH ₂ PO ₄ 0.33, CaCI ₂ 1.2, MgCI ₂ 1.0, Hepes 10; pH adjusted to 7.4 with NaOH).

Obtaining cells isolated from rabbit ventricular cardiomyocytes

Isolated ventricular cells are obtained from hearts of New Zealand maize rabbits as described in A. d'Anglemont de Tassigny et al., Fund Clin Pharmacol, 18: 531-38, 2004. In short, rabbits (2, 0-2.5 kg) are anesthetized with a solution of pentobarbital (50 mg / kg) and then receive heparin (200 lU / kg). The hearts are excised and immediately perfused, for 10 to 15 minutes using a Langendorff apparatus without recirculation with an isotonic solution of oxygenated tyrode (without calcium) (95%, 2-5% CO ₂ ) (in mM: NaCI 135, KCI 5.4, Na ₂ PO ₄ 0.33, MgCI ₂ 1.0, HEPES 10, pH adjusted to 7.4 with 1 N NaOH at 37 ° C, 280-300 mOsmol / kgH ₂ O). Then, all the hearts are perfused for 3 minutes in "recirculation" mode with the same Tyrode solution without calcium (coronary flow, 10-15 ml / min) added with 1 mg / ml of collagenase type II and 0.28 mg / mL of protease type XIV. Finally, all the hearts are perfused in non-recirculation mode with the same Tyrode solution supplemented with 0.3 mM CaCl2 for 10 min. The left ventricle is removed and cut into small pieces, cell dissociation is achieved by gentle mechanical agitation. Extracellular calcium is added incrementally every 15 minutes to arrive at a physiological concentration of 1.0 MM. The isolated myocytes are maintained in a serum-free medium containing (in mM) NaCI 110, KCI 5.4, Na ₂ PO ₄ 0.33, NaHCO ₃ 25, Glucose 5, MgCI ₂ 0.8, CaCI ₂ 1, pH adjusted at 7.4 until 1.5 hours before the experiment. All of the cells are baguette-shaped, have clear cross striation and do not have a vesicle on their surface under an optical microscope.

Annexin V labeling

The annexin V labeling of phosphatidylserine was used as a quantitative method of measuring apoptosis using the MiniMacs cell isolation kit (Miltenyi Biotec, Bergisch, Gladbach, Germany). In short, the cells exhibiting phosphatidylserine are magnetically labeled with annexin V microbeads, then passed through a column placed in a field magnetic. Labeled cells (which have magnetically labeled phosphatidylserine) are retained in the column while unlabeled cells (necrotic and non-apoptotic cells) are not retained. The column is removed from the magnetic field, the cells exposing magnetically retained phosphatidylserine are eluted as a positive fraction and counted with a Mallassez cell. The percentage of apoptotic cells is then related to the initial number of cells. Measuring caspase-3 activity

The caspase-3 activity is used as a quantitative method for measuring apoptosis. Briefly, the cells are lysed and the supernatant is used for the measurement of caspase-3 activity using the kit AK-005 (Biomol Research Laboratories, Plymouth Meeting, PA, USA). The fluorogenic substrate for measuring caspase-3 activity (DEVD) is labeled with the fluorochrome 7-amino-4-methyl coumarin (AMC) which produces a yellow-green fluorescence detectable in UV light at 360/460 nm for 210 min . AMC is released from the substrate by cleavage by caspase-3, the expression of the enzyme is expressed in fmol / min. Measure of contractility

The myocytes are transferred to a chamber at 37 ° C continuously perfused and positioned on the stage of an inverted microscope. The chamber is perfused with physiological buffer containing (in mM): NaC1 140; KCI 5.4; CaCl ₂ 1; MgCl ₂ 0.8; HEPES 10 and glucose 5.6 (pH = 7.4; 290 mOsmol / kgH ₂ O). Myocyte contraction is induced once per second (1 Hz) with platinum field electrodes placed in the chamber and connected to a stimulator. The images are continuously captured with a 20x objective and transmitted to a CCD camera at the rate of 240 samples / s. The CCD camera images are projected onto a video screen.

The myocytes were selected for the study according to the following criterion: a baguette-like appearance with very visible striations and no intracellular vacuole, no spontaneous contraction when stimulated with 1 mM Ca ²⁺ , and with a length constant rest and amplitude of contraction. The length of the sarcomeres was measured using a video image analysis program and the data was captured at a rate of 240 samples / s. Camera images have been converted to length of sarcomere. The percentage of contraction is calculated from these data over the length of the sarcomere.

Data analysis

All data were expressed as mean ± standard deviation. Data comparisons between the different groups were carried out by ANOVA followed by a Student test with a significant difference at p <0.05.

> Experimental protocol

Apoptosis is induced in isolated cardiomyocytes by exposure for 3 to 8 hours to 1 μM of doxorubicin added in an isotonic solution containing (in mM) NaCI 110, KCI 5.4, Na ₂ PO ₄ 0.33, NaHCO ₃ 25, Glucose 5, MgCl ₂ 0.8, CaCl ₂ 1, pH adjusted to 7.4. The labeling with annexin V was carried out 3 hours after the start of exposure to doxorubicin since this phenomenon appears very early in the apoptotic cascade. Measurements of caspase-3 activity are carried out 8 h after exposure to doxorubicin since this phenomenon takes place later in the phenomenon of apoptosis. Cardiomyocyte contractility was measured every hour during the 8 hours of exposure to doxorubicin. After all the treatments, the cells were compared to the control cardiomyocytes not exposed to doxorubicin.

The cardiomyocytes were pretreated with the compound 3,5-seco-4-nor-cholestan-5-one oxime-3-ol for 15 min before exposure to doxorubicin. Two concentrations of this compound were tested during this study: 0.1 and 0.3 μM.

> Results

The mean length of the cell sarcomeres used in this study was not significantly different between the groups. o Effect of doxorubicin on myocyte contractility and apoptosis Exposure to doxorubicin resulted in a reduction over time of the shortening of the sarcomere. The shortening of the peak with doxorubicin was similar to control during the first three hours and then became significantly reduced after 4 h of exposure (-53.20 ± 7.70% against - 19.49 ± 2.06% compared to the line baseline of doxorubicin and control respectively, p <0.05, n = 5). Treatment with 1 μM doxorubicin induced apoptosis with a significant increase in labeling with annexin V and in caspase-3 activity. o Effect of 3,5-seco-4-nor-cholestan-5-one oxime-3-ol on dysfunction at the level of contractility induced by doxorubicin and on apoptosis.

Treatment with 1 μM doxorubicin resulted in a significant reduction in the shortening of the peak of ventricular cardiomyocytes which is abolished in the presence of 3,5-seco-4-nor-cholestan-5-one oxime-3-ol (0,1 and 0.3 μM). Indeed, after 4 h of exposure, the shortening of the peak under doxorubicin (-53.20 ± 7.70%) became significantly reduced with the compound to 0.1 μM (-18.9 ± 5.4%) and 0.3 μM (-8.1 ± 9.6%) from the baseline.

In addition, increases in annexin V labeling and caspase-3 activity due to doxorubicin were blocked by 3,5-seco-4-nor-cholestan-5-one oxime-3-ol at 0.1 and 0.3 μM.

Apoptosis evaluated as a% change in labeling with annexin V 3 hours after doxorubicin gives the following results: control: 100%; doxorubicin: 320% ± 48.7; doxorubicin + 3,5-seco-4-nor-cholestan-5-one oxime- 3-ol 0.1 μM: 116.3% ± 15.1; doxorubicin + 3,5-seco-4-nor-cholestan-5-one oxime- 3-ol 0.3 μM: 137.3% ± 19.3. The results for the caspase-3 activity measurements are as follows: control: 19 + 9 fmol / min; doxorubicin: 120 ± 15 fmol / min; doxorubicin + 3,5-seco-4-nor-cholestan-5-one oxime- 3-ol 0.1 μM: 27 ± 20 fmol / min; doxorubicin + 3,5-seco-4-nor-cholestan-5-one oxime- 3-ol 0.3 μM: 15 ± 7 fmol / min. > Comments and conclusions

The compound 3,5-seco-4-nor-cholestan-5-one oxime-3-ol shows a cardioprotective effect on the contractility dysfunction induced by doxorubicin and on apoptosis on isolated rabbit cardiomyocytes. The molecule, when used in appropriate doses can effectively protect against doxorubicin-induced cardiotoxicity which is known to be the limiting factor in the treatment of cancer patients with this anthracycline. Thus, the compound 3,5-seco-4-nor-cholestan-5-one oxime-3-ol could be used to limit the cardiotoxicity of doxorubicin in these patients. Example 13 Effect of the compounds 3,5-seco-4-nor-cholestan-5-one oxime-3-ol and 3,5-seco-4-nor-choIestan-5-one oxime-3-N, N- dimethylglycine ester in an in vivo model of myocardial infarction in mice

The aim of this experiment is to study in vivo in a model of coronary occlusion-reperfusion in mice, the cardioprotective properties of the 3,5-seco-4-nor-cholestan-5-one oxime-3-ol compounds and 3,5-seco-4-nor-cholestan-5-one oxime-3-N, N-dimethylglycine ester.

The test compounds are administered intravenously to mice 5 minutes before reperfusion of the previously ischemic myocardium. As controls, the vehicles of the compounds, respectively beta-cyclodextrin (hereinafter called DCD) and water, are administered under the same conditions as the compounds.

In order to determine the effect of the compounds tested, the size of the infarction is measured after a 24 hour reperfusion.

Animal surgical instrumentation

Male C57BL6 mice aged 6 to 8 weeks are anesthetized with sodium pentobarbital (50 mg / kg ip) and are ventilated after intubation with a mixture of O2 / CO ₂ (95% / 5%) throughout the experiment . The surface electrocardiogram (ECG) is recorded and viewed on an oscilloscope for the duration of the surgery. Under rigorous aseptic conditions, the jugular vein is catheterized for intravenous administration of the compounds. A left lateral thoracotomy is performed and after pericardiotomy, a major branch of the left coronary artery is located in the latero-posterior region of the left ventricle. A prolene wire number 8 is positioned around this artery so as to form a mobile loop for the realization of a temporary coronary occlusion.

Experimental protocols

All mice underwent temporary coronary occlusion for 30 minutes. Ischemia of the myocardial region is confirmed by the presence of cyanosis of the myocardial surface and by a deviation of the ST segment of the EKG. Each of the compounds (treated group, n = 10) or its solvent (control group, n = 10) was administered as a bolus intravenously 5 min before the lifting of the 30 min of coronary occlusion.

3,5-seco-4-nor-cholestan-5-one oxime-3-ol, previously dissolved at a final concentration of 0.46 mg / ml in a solution of DCD at 30% in phosphate buffer prepared by saturation followed by centrifugation, is administered at a dose of 1 mg / kg.

3,5-seco-4-nor-cholestan-5-one oxime-3-N, N-dimethylglycine ester, previously dissolved at a final concentration of 1.56 mg / ml in sterile water by shaking and sonication is administered at a dose of 3.9 mg / kg.

The same volume of vehicle is administered in the corresponding control groups.

The reperfusion was confirmed by the appearance of the QRS segment of the EKG.

After closing the chest plane by plane and evacuating the pneumothorax by aspiration using a drain, the mice are then gradually awakened and weaned from their ventilatory assistance until resumption of normal spontaneous ventilation. If necessary, buprenorphine (1 mg / kg) is administered intraperitoneally to provide effective pain relief coverage.

Twenty-four hours after the end of the coronary occlusion, the mice are re-anesthetized with sodium pentobarbital (50 mg / kg i.p.) and heparin is administered intravenously (heparin Choay © 1000IU / kg). A ligation of the coronary artery is performed in the same place as that which was the subject of the first occlusion 24 hours previously. The mice are then euthanized by a lethal dose of saturated potassium chloride and their heart is rapidly excised and then mounted by the aortic artery on a retrograde perfusion system of the Langendorf type.

A 5% Evans blue solution is retrograde perfused so that the healthy myocardium is colored blue; the area made ischemic during coronary occlusion, or "area at risk AR" remaining uncolored by default. The left ventricle is then cut into 5 slices of equal thickness (1 mm) using a slicer specially designed for mouse hearts (Les Isolants de Paris, Palaiseau) which are then weighed.

These slices are incubated in a solution of triphenyltetrazolium chloride (TTC, Sigma, Poole, UK) at 1% pH 7.4 for 20 min at 37 ° C and then fixed in 4% formalin. The TTC has the property of coloring the non-infarcted myocardium in red, and therefore showing the infarcted areas in white. Each heart slice is placed under a stereo microscope for taking a high definition digital photo.

The quantification of the infarction and the area at risk is carried out by planimetry (Scion Image, Scion, Frederick MD, USA) and related to the weight of each slice.

The risk area (non-blue zone) is expressed as a percentage of the weight of the left ventricle. Heart attack sizes are expressed as a percentage of the weight of the area at risk.

All values of infarction size and risk area are expressed as mean ± SEM. A one-factor ANOVA followed by a non-even Student's t-test with Bonferonni correction is used to compare the areas at risk and the sizes of infarction between the experimental groups, the significance level being fixed at p <0.05 .

Results and conclusions

The infarction sizes of the mice treated with the compounds differ significantly from those of the mice treated with the vehicle.

The results presented in the table below are expressed on the one hand by the area at risk and on the other hand by the size of the infarction.

In the experimental model used, the two compounds tested reduce the size of myocardial infarction. In addition, the results showed that the compounds reduce the sizes of infarction regardless of the extent of the risk zone.

These results therefore show that these two compounds exhibit cardioprotective effects in vivo.

Example 14: Effect of the compound 3,5-seco-4-nor-cholestan-5-one oxîme-3-ol in an in vivo model of acute hepatotoxicity.

In this experiment, the capacity of the compound 3,5-seco-4-nor-cholestan-5-one oxime-3-ol is tested to protect the hepatocytes.

Hepatocytes, like many other cells, carry the Fas / CD95 receptor on their cytoplasmic membrane. Stimulation of this Fas pathway induces cell death by activating the caspase cascade.

An acute pattern of liver damage can be induced by a single injection of the anti-Fas Jo2 antibody (Ogasawara et al., Nature, August 1993), producing severe liver damage and resembling viral, autoimmune or hepatitis-induced hepatitis. drugs.

Alanine Aminotransferase (ALAT) also called Serum Glutamic Pyruvic Transaminase (SGPT) is an enzyme found in hepatocytes. Its activity increases significantly in plasma after hepatic lysis and is therefore a good marker for assessing liver damage. Animal Materials and Methods

Adult male CD1 mice from "Elevage Janvier" (Le Genest-Saint-Isle, France) were used. The animals were identified individually and had free access to water and food.

The installations were maintained on a controlled light cycle (7:00 - 19:00), and at temperatures of 20 ± 2 ⁰ C and humidity of 50 ± 20%.

Preparation of the Jo2 antibody

A stock solution of hamster monoclonal antibody CD95 (Fas), called Jo2, from Pharmingen (BD Biosciences, ref. 554254 batch 32699) is prepared at a concentration of 1 mg / ml in water. The dilutions used are made in 0.9% sodium chloride in water.

Preparation of the compound to be tested

The desired amount of 3,5-seco-4-nor-cholestan-5-one oxime-3-ol is weighed and ground into a fine powder, then mixed with Cremophore EL (Sigma C5135) and absolute ethanol ( Carlo Erba RPE 41571) (respectively 5% and 10% of the final volume). After complete dissolution, 0.9% sodium chloride in water is added immediately (85% of the final volume).

Two types of experiments are conducted: intoxication by Jo2 followed by a dosage of ALT and lethal intoxication by Jo2. intoxication by Jo2 and dosage of ALT:

Protocol

A pretreatment with 3,5-seco-4-nor-cholestan-5-one oxime-3-ol is carried out at doses of 10 and 30 mg / kg by intraperitoneal administration 1 hour before the administration of the antibody Jo2. The Jo2 antibody is administered by intraperitoneal injection at a dose of 125 μg / kg in a volume of 5 mL / kg of body weight.

A control is carried out on animals receiving a pretreatment by intraperitoneal administration 1 hour before the administration of the antibody of an identical volume of solution having been used for the preparation of the test compound, without compound. Dosage of ALT

Blood from anesthetized mice is taken 24 hours after administration of Jo2. The dosage of ALT is carried out using a kit (Roche Diagnostics) with a spectrophotometer (Hitachi Modular), according to the method standardized by NFCC (International Federation of Clinical Chemistry).

Results and conclusions

The intraperitoneal administration of Jo2 at 125 μg / kg does not cause mortality in the mice within 24 hours of the injection.

The ALAT activity is significantly reduced by the compound 3,5-seco-4-nor-cholestan-5-one oxime-3-ol at 10 and 30 mg / kg.

Table 1: ALT activity measured 24 hours after administration of Jo2

^** : p <0.01, ANOVA followed by Dunnett's comparative test compared to the placebo group

3,5-seco-4-nor-cholestan-5-one oxime-3-ol administered at 10 and 30 mg / kg, 1 hour before the Jo2 antibody, made it possible to limit the cell death induced by a sublethal dose d 'antibody.

The ALAT ₁ activity, biomarker of hepatic cytolysis in plasma, is significantly lower in mice treated with 3,5-seco-4-nor-cholestan-5-one oxime-3-ol than in non-control mice processed. Lethal poisoning by the Jo2 antibody

In this experiment, the effect of 3,5-seco-4-nor-cholestan-5-one oxime-3-ol on the survival of the animals after administration of a lethal dose of the antibody Jo2 is evaluated.

Protocol

The Jo2 antibody is administered by intraperitoneal injection at a dose of 200 or 250 μg / kg in a volume of 5 mL / kg of body weight. 3,5-seco-4-nor-cholestan-5-one oxime-3-ol is tested at 10 and 30 mg / kg, as a pretreatment, 1 hour before the administration of Jo2 or in post-treatment, 1 hour after l administration of Jo2.

A control is carried out on animals receiving a pretreatment by intraperitoneal administration 1 hour before or 1 hour after the administration of the antibody of an identical volume of solution having been used for the preparation of the test compound, without compound.

Results and conclusions

Table 2: Survival of animals at 24h

At the doses administered, the Jo2 antibody induces significant mortality within 24 hours (70 to 100% of the animals) in the control group.

Pre-treatment and / or post-treatment with 3,5-seco-4-nor-cholestan-5-one oxime-3-ol at the doses administered induces an increase in the survival of the animals.

Thus, when a lethal dose of antibody is used (200 or 250 μg / kg), 3,5-seco-4-nor-cholestan-5-one oxime-3-ol, administered at 10 and 30 mg / kg, 1 hour before or after the antibody, increases the survival of the mice over 24 hours.

Conclusions

The acute hepatotoxicity model induced in mice by an anti Fas antibody (Jo2) made it possible to demonstrate the hepatoprotective properties of 3,5-seco-4-nor-cholestan-5-one oxime-3-ol.

These remarkable effects make it possible to envisage for the compounds of formula I a use in the preparation of a cytoprotective medicament in general.

Claims

1. Use of at least one compound corresponding to formula I

in which

- X and Y taken together represent a keto function (= 0), an oxime group (= NOH) or a methyloxime group (≈NHOMe) where X represents a hydroxyl and Y a hydrogen atom;

- B represents a hydroxyl radical and C and D, identical or different, represent a hydrogen atom, or an alkyl radical, linear or branched, comprising from 1 to 4 carbon atoms; or B and C taken together represent a keto function and D a methyl, hydroxyl or methylamine radical; or B and C represent a hydrogen atom and D a methylamine radical; or B and C taken together represent an oxime group and D a methyl radical; and R represents an alkyl radical, linear or branched, comprising from 1 to 10 carbon atoms; or one of its addition salts with pharmaceutically acceptable acids, or one of its esters or one of the addition salts with pharmaceutically acceptable acids of said esters, for obtaining a cytoprotective medicament, except for a neuroprotective drug.

2. Use according to claim 1, characterized in that in formula I, R represents the cholestane radical of formula II

3. Use according to any one of claims 1 or 2, characterized in that in formula I, X and Y taken together represent a keto function.

4. Use of a compound according to any one of claims 1 to 3, characterized in that in formula I, B represents a hydroxyl radical and C and D, identical or different, represent a hydrogen atom or, a alkyl radical, linear or branched, comprising from 1 to 4 carbon atoms.

5. Use according to any one of claims 1 to 3, characterized in that in formula I, B and C taken together represent a keto function and D represents a methyl radical.

6. Use according to any one of claims 1 or 2, characterized in that in formula I, X and Y taken together represent an oxime group.

7. Use according to claim 1, characterized in that the compound of formula I is chosen from 3,5-seco-4-nor-cholestan-5-one oxime-3-ol, 3,5-seco-4 -nor-cholestan-5-one oxime-3-methyl alcohol, or 3,5-seco-4-nor-cholestan-5-one oxime-3-dimethyl alcohol, or one of its addition salts with pharmaceutically acceptable acids, or one of its esters or one of the addition salts with pharmaceutically acceptable acids of said esters.

8. Use according to any one of the preceding claims, characterized in that the medicament is intended for the treatment or prevention of necrosis and / or pathological apoptosis and / or necroptosis (antinecrotic and / or antiapoptotic and / or antinecroptotic drugs) or conditions such as diseases of the bones, joints, connective tissue and / or cartilage; muscle diseases; skin diseases; cardiovascular illnesses ; circulatory diseases; hematological and vascular diseases; lung diseases; diseases of the gastrointestinal tract; liver disease; diseases of the pancreas; metabolic diseases; kidney disease; viral and bacterial infections; severe poisoning; degenerative conditions associated with Acquired Immune Deficiency Syndrome (AIDS); disorders associated with aging; inflammatory diseases; autoimmune diseases; dental disorders; ophthalmic diseases or disorders; auditory pathway diseases; diseases associated with mitochondria.

9. Use according to any one of the preceding claims, characterized in that the medicament is intended for the protection of cardiac cells (cardioprotective medicament).

10. Use according to any one of claims 1 to 9 characterized in that the medicament is intended for the protection of hepatic cells (hepatoprotective medicament).

11. Use according to any one of claims 1 to 9, characterized in that the medicament is intended for the treatment or prevention of diseases associated with mitochondria.

12. Use according to any one of claims 1 to 9, characterized in that the medicament is intended for the protection of cells, of a tissue or of an organ, before, during or after a transplant.

13. Composition, in particular pharmaceutical or drug composition, comprising at least one compound of formula I as described in claim 1, or one of its addition salts with pharmaceutically acceptable acids or one of its esters or one addition salts with pharmaceutically acceptable acids of said esters.